Hepatitis B is a worldwide health problem. Our long term objective is to develop hairpin Ribozyme (Rz) gene therapy for the treatment of HBV infection. We have successfully completed both Specific Aims of our Phase I SBIR grant. In addition, we have modified two of the best ribozymes and evaluated their anti-viral effects in cells. The structurally modified ribozymes inhibited HBV production by at least 83%. These encouraging results indicate the feasibility of ribozyme-mediated gene therapy for the treatment of HBV infections. The work from the Phase I SBIR studies has been accepted for publication In GeNe Therapy (in press). Therefore, we further propose a Phase II SBIR study with the following aims: 1) to further optimize the two effective anti-HBV ribozymes (BR1-TL and BR3-TL) by varying the helix l length and kinetically characterizing the resulting variants to identify the ones with the greatest catalytic effects: 2) to generate viral vectors (adenoviral vectors and adeno-associated viral vectors) that express the single most active ribozyme genes identified and to generate vectors expressing both ribozymes in the same vector construct; and 3) to evaluate vector-mediated ribozyme activity in reducing HBV production in liver cell culture and evaluating pre-clinical efficacy and safety in a SCID-Hu mouse model. Achieving these Aims will generate the most effective ribozyme viral vector(s) to be used for a phase I clinical trial for treatment of HBV infection. PROPOSED COMMERCIAL APPLICATION: Immusol plans to commercially develop a Rz gene therapy approach to HBV infection. We will manufacture and provide Rz-expressing viral vectors in a vial. The vectors will then be provided to physicians around the world; they will carry out the in vivo intravenous vector-mediated gene transfer into patient liver cells to combat HBV virus.